Our understanding of the basic mechanisms of mRNA expression has been instrumental in the identification of molecular mechanisms mediating posttranscriptional control of gene expression, which led to the development of RNA optimization (also referred to as codon optimization) as a key strategy to improve gene expression, a method which is presently a key technology for many gene therapy applications including therapeutic HIV vaccines and cytokine DNAs which are used in this project. The use of cytokine pDNAs (IL-12, IL-15) as molecular vaccine adjuvants was found to improve the quantity and alter the quality of the immune responses. To optimally use these cytokines, we found that IL-15/IL-15Ra (hetIL-15) as well as the IL-12 cytokine family use similar highly regulated steps posttranscriptional and posttranslational regulation strategies. We reported that coexpression of IL-15 and IL-15Ra from the same cell is critical for the production of hetIL-15 and analyzed its complex glycosylation pattern. hetIL-15, as a lymphocyte growth factor, represents a promising candidate in HIV immunotherapies against HIV and cancer. HetIL-15 has the potential that harness the patient's own immune system, inducing proliferation of NK and T cells, and to induce CTL to reduce or clear the HIV reservoir and, similarly, as part of cancer immunotherapy to induce anti-tumor CTL. Thus, HIV and cancer immunotherapy share underlying immunologic features and our studies using cytokine therapy approach may shed light towards the cure of both. To test applications of hetIL-15 in immunotherapy against cancer and HIV, we produced hetIL-15 from mammalian cells. HetIL-15 is presently in clinical trials for immunotherapy of metastatic cancers. We have further shown that hetIL-15 is able to replace the need for lymphodepletion prior to Adoptive Cell Transfer (ACT) for cancer therapy. hetIL-15 induces lymphocyte entry into tumor sites and activation of intratumoral CD8+ T cells. In contrast to other protocols, hetIL-15 induces an enrichment of antigen specific lymphocytes in the tumor over time and greatly increases CD8+/Treg cell ratio. Application of hetIL-15 to ACT will provide new tools and techniques for cancer immunotherapy protocols. Elimination of the need for lymphodepletion will make more patients eligible for cell transfer protocols. In addition, IL-15 treatment could be a general method to place T cells into tumors, increasing the success rate of other immunotherapy interventions. In another line of research, we produced and developed the extracellular vesicle (EV) technology to deliver hetIL-15 as immunotherapy to tumor sites. Our procedure have the potential to be be readily applied to generate purified preparations of engineered EV for research and development. To use IL-12 DNA to its full potential, we studied the biology of this glycosylated 70 kDa heterodimeric cytokine to maximize cytokine production. Although the production of each subunit is independently regulated, coexpression of both molecules in the same cell is essential to form biologically active heterodimer. Prompted by our findings on the critical intracellular regulatory step of IL-15/IL15Ra cross-stabilization, we investigated the posttranscriptional regulation and interaction of the p35 and p40 subunits leading to optimal IL-12p70 production. Investigating molecular steps controlling IL-12p70 biosynthesis, we found that the combination of RNA-optimized gene sequences, and importantly, fine-tuning of the relative expression levels of the two subunits within a cell resulted in greatly increased production of the IL-12p70 heterodimer. Importantly, we discovered that p40 enhances the p35 stability and promotes its intracellular trafficking, resulting in formation of a stable, efficiently secreted IL-12p70 complex. Dual expression plasmids for IL-12p70 were designed to obtain favorable relative levels of the two subunits and optimal IL-12 expression used as adjuvant for our DNA vaccine studies. Like IL-12, the related IL-23 and IL-27 are similarly regulated and cytokine production is controlled by the p40 and EBI3 chains, respectively. These cytokine pDNAs provide important molecular tools to be tested as molecular adjuvants in vaccine and in cancer immunotherapy, with promising future translational applications. Towards this, we further developed high expresser mammalian cell lines grown under defined serum-free conditions. We reported an efficient one-step purification of IL-12 protein without affinity tag. Using the purified IL-12 cytokine, we now demonstrated for the first time that heparin serves as a co-receptor capable of maintaining a reservoir of IL-12 at the cell surface and, thus, enhancing its activity. Our understanding of the molecular biology of these cytokines provided the critical information necessary for efficient cytokine production. Despite great progress in understanding the immunobiology of HIV infection and AIDS pathogenesis, the development of a therapeutic HIV vaccine has not been achieved. HIV vaccine development is complicated by virus variability, the nature of the viral antigens, immune dominance and the short persistence of vaccine-induced humoral immunity at mucosal sites. As described in project 1, we developed pDNA-based vaccines to induce robust cytotoxic T cell responses to the conserved elements (CE) of HIV-1 Gag, selecting regions by stringent conservation, functional importance, independent of 'protective' haplotypes associated with virus control, and association with immune control in HIV-infected LTNP. We demonstrated that a vaccine comprising conserved elements (CE) of HIV-1 p24gag as prime followed by a combination of CE+gag pDNA as boost elicits broad, potent and durable T cell responses targeting these conserved regions, efficiently overcoming the dominance imposed by the Gag variable regions. Similar to the HIV CE DNA vaccine, we have developed SIV CE DNA by analogy and found that vaccination with p27CE pDNA was also critical for the efficient induction and increased the frequency of Ag-specific T cells with cytotoxic potential (granzyme B+ CD107a+) targeting subdominant CE epitopes, compared with the responses elicited by the p57gag pDNA vaccine. Following p27CE pDNA priming, the CE+gag pDNA booster vaccination elicited significantly broader CE epitope recognition, and thus, a more profound alteration of the immunodominance hierarchy. Hence, SIV/HIV vaccine regimens comprising CE pDNA prime and CE+gag pDNA booster vaccination significantly increased cytotoxic T cell responses to subdominant highly conserved Gag epitopes and maximized response breadth. We are using the CE DNA vaccine regimen in SIV infected in ART-treated macaques and found that it induced robust cytotoxic T cell response. Combination of the HIV CE vaccine with hetIL-15 treatment is currently under evaluation as potent immunotherapeutic approach against SIV. As a collaborative effort, we have also developed and clinical trial in HIV-infected persons under HAART (A5369) to test the immunogenicity of the HIV CE vaccine regimen (opens end 2017). Both trials, HVTN 119 in HIV naive persons and A5369 will be evaluated using the techniques in the same lab. This will allow us to directly compare and contrast the CE immunogenicity under different circumstances.